US 2011 (02818O2A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/028.1802 A1 Armbruster et al. (43) Pub. Date: Nov. 17, 2011
(54) COMPOSITION FORTREATMENT AND Publication Classification PREVENTION OF HARLOSS AND (51) Int. Cl. PREMATURE GRAYING OF HAR A638/08 (2006.01) Inventors: Franz Paul Armbruster, Bensheim GOIN 33/53 (2006.01) (75) A61O 5/00 (2006.01) (DE); Ralf Paus, Lubeck (DE): C07K 6/26 (2006.01) Dorothee Langan, Osnabruck (DE) A638/22 (2006.01) (73) Assignee: Immudiagnostik AG, Bensheim A61O 700 (2006.01) (DE) (52) U.S. Cl...... 514/12.9, 514/9.7:530/389.2: (21) Appl. No.: 12/745,247 435/7.1 (22) PCT Fled: Dec. 1, 2008 (86) PCT NO.: PCT/EP2008/066542 (57) ABSTRACT Composition for treatment and prevention of human hair loss S371 (c)(1), and/or hair greying, comprising an effective amount of thy (2), (4) Date: Aug. 27, 2010 mic peptides of the family thymulin, thymosin alpha-1 and (30) Foreign Application Priority Data thymosin beta-4 and pharmaceutical excipient, diluent or car rier. Method and pharmaceutical composition for treatment Nov.30, 2007 (EP) ...... O7122054.5 and prevention of balding, hair loss, and alopecia. Patent Application Publication Nov. 17, 2011 Sheet 1 of 13 US 2011/028.1802 A1
FIG. 1
-Control (Culture medium only) -Thymulin 10pg/ml -Thymulin 100pg/ml -Thymosino.1 100ng/ml -Thymosino.1 1000ng/ml -Thymosin 34 100ng/ml -Thymosin 34 1000ng/ml
FIG. 2
OOOOOO -Control (Culture medium only) OOOOOO -Thymulin 10pg/ml OOOOOO -Thymosin 34 100ng/ml OOOOOO -Thymosin 34 1000ng/ml
FIG. 3
() () OOOOOO -Control (Culture medium only) -Thymulin 10pg/ml
) () OOOOOO OOOOOO -Thymulin 100pg/ml -Thymosin a 1 100ng/ml CD OOOOOO -Thymosin a 1 1000ng/ml 5 5 OOOOOO -Thymosin 34 100ng/ml -Thymosin (34 1000ng/ml Patent Application Publication Nov. 17, 2011 Sheet 2 of 13 US 2011/028.1802 A1
FIG. 4A
- Assay #1 - Thymulin
120,00 100,00 |- 80,00 60,00 40,00 20,00 0.00 -- Control --Thymulin 10pg/ml Thymulin 100pg/ml
FIG. 4B
Assay #1 - Thymosin alpha1 -0- Control
g -H-Thymosin alpha1
- 100ng/ml
Thymosin alpha1 1000ng/ml Patent Application Publication Nov. 17, 2011 Sheet 3 of 13 US 2011/028.1802 A1
FG, 4C
ASSay #1 - thymosin beta4
120,00 100.00 80,00 60,00 40,00 20,00 0.00 -- Control -- Thymosin beta4 100ng/ml Thymosin beta4 1000ng/ml
F.G. 5
Assay #2 - Thymulin and Thymosin beta4
70,00 60,00 50,00 40,00 30,00 - 20,00 1 O,OO O,OO -- Control --Thymulin 10pg/ml
- Thymosin beta4 100ng/ml ->{-Thymosin beta4 1000ng/ml ) Patent Application Publication Nov. 17, 2011 Sheet 4 of 13 US 2011/028.1802 A1
FIG 6A
Assay #3 - Thymulin
120 100 80
60 40 20 O -- Control --Thymulin 10pg/ml Thymulin 100pg/ml
FIG. 6B
- Assay #3 - Thymosin alpha1
100 80 60 40 a. p. 0.017 20 b. p=0.023 O -0- Control -H Thymosin alpha1 100ng/ml Thymosin alpha1 1000ng/ml J Patent Application Publication Nov. 17, 2011 Sheet 5 of 13 US 2011/028.1802 A1
FIG. 6C
—Assay — #3 - Thymosin— beta4 —-
100 90 80 70 60 50 40 30 20 10 4. O -0- Control --Thymosin beta4 100ng/ml Thymosin beta4 1000ng/ml
FIG. 7
Patent Application Publication Nov. 17, 2011 Sheet 6 of 13 US 2011/028.1802 A1
F.G. 8A
D late Catagen Omid Catagen early Catagen O Anagen
O late Catagen Omid Catagen Eg early Catagen Anagen
Control Thymulin Thymosin Thymosin 10pg b4 100ng b4 1000ng Patent Application Publication Nov. 17, 2011 Sheet 7 of 13 US 2011/028.1802 A1
F.G. 8C
O late Catagen O mid Catagen early Catagen O Anagen
sis is sis
F.G. 9
Mean percentage of proliferating cells (after 9 days of culture, Anagen only)
Patent Application Publication Nov. 17, 2011 Sheet 8 of 13 US 2011/028.1802 A1
FIG. 10
FIG 11
Patent Application Publ Ca tion NOV. 17 2011 Sheet 9 Of 13 US 2011/028.1802 A1
FIG. 13
FIG 14
FIG. 15 Patent Application Publication Nov. 17, 2011 Sheet 10 of 13 US 2011/028.1802 A1
Š Š. 8 S Š S
FIG. 18
Patent Application Publication Nov. 17, 2011 Sheet 11 of 13 US 2011/028.1802 A1
FIG. 20 A
Patent Application Publication Nov. 17, 2011 Sheet 12 of 13 US 2011/028.1802 A1
FIG. 22 A
FIG.22 B
FIG. 22 C
Patent Application Publication Nov. 17, 2011 Sheet 13 of 13 US 2011/028.1802 A1
FIG. 23 A
FIG. 23 B
US 2011/028.1802 A1 Nov. 17, 2011
COMPOSITION FOR TREATMENT AND beta-Thymosins, small acidic peptides with multiple func PREVENTION OF HAIR LOSS AND tions; Intern. J Biochem & Cell Biol 2001, 33: 205-220). PREMATURE GRAYING OF HAIR Numerous biological effects have been reported for this pep tide but best known are its inhibition of G-actin polymeriza FIELD OF THE INVENTION tion and the stimulation of cell migration (Huff T et al., beta-thymosins, small acidic peptides with multiple func 0001. The invention relates to compositions for treatment tions; Intern. J Biochem & Cell Biol 2001, 33: 205-220). and prevention of hair loss and premature greying of hair. Thymosin beta-4 has been found to stimulate wound healing. decrease inflammation reactions and increase re-epitheliali BACKGROUND OF THE INVENTION sation and angiogenesis (Malinda KMetal. Thymosin beta4 0002. The thymus was not a well-understood organ until accelerates wound healing; J Invest Dermatol. 1999, 113: studies in the 1960's that animals whose thymus organs were 364-8, Philp D et al. Thymosin beta4 promotes angiogenesis, removed, developed profound immune deficiencies and died. wound healing, and hair follicle development, Mech Aging Ever since the thymus is known to be responsible for the and Develop 2004, 125:113-115). Thymosin beta-4 is development and regulation of T cell immunity. The thymus expressed in hair follicle stem cells in a cycle-related fashion seems to exert its regulatory functions through the secretion and was noted to stimulate hair growth in rats (Philp D et al. of various non-cellular, hormone-like products, called thymic Thymosin beta4 increases hair growth by activation of hair peptides. follicle stem cells: The FASEBJ Express Article doi:10.1096/ 0003) Thymulin is a nonapeptide, which is active when f.03-0244fe published online Dec. 4, 2003). WO 03/063775 coupled with zinc and which plasma levels follow a circadian discloses the use of thymosin beta-4 and peptides thereof for rhythm, peaking in the early hours of the morning (Safieh the promotion of hair growth and treatment of allopecia. Garabedian et al., Thymulin and its role in immunomodula 0006. It is clear that thymic peptides are powerful biologi tion; J. Autoimmunity 1992. 5:547-555; Mocchegiani et al., cal mediators. Notwithstanding, the full extent of effects of Presence of links between zinc and melatonin during the thymic peptides is still not known. Consequently, the state of circadian cycle in old mice: effects on thymic endocrine activ the art represents itself as a problem and it is an object of the ity and on survival; J. Neuroimmunology 1998, 8.6:111-122). invention to provide further compositions and therapeutic Recent research showed that thymulin acts favourably in applications on the basis of thymic peptides. patients with viral diseases, chronic pain and cancer. Thus, thymulin is suspected to play a role in the communication SUMMARY OF THE INVENTION between the neuroendocrine system and the immune system. 0007. The present invention provides a pharmaceutical Thymulin also affects the release of ACTH, PRL, GH, TSH composition for treatment and prevention of hair loss and and LH from the pituitary gland (Hadley et al., Thymulin premature human hair greying, comprising an effective stimulates corticotrophin release and cyclic nucleotide for amount of thymic peptides, in particular thymulin, thymosin mation in the rat anterior pituitary gland; Neuroimmuno alpha-1 (TA1) and thymosin beta-4 (TB4). modulation 1997, 4:62-9: Brown OA et al., Thymulin stimu 0008. In one embodiment of the invention, the pharmaceu lates prolactin and thyrotropin release in an age-related tical composition comprises an effective amount of thymulin manner: Mechanisms of Aging and Development 1998, 104: and/or thymosin alpha-1 for treatment of hair shedding and 249-62; Brown O Aetal, Growth hormone-releasing activity telogen effluvium by catagen inhibition. Further applications of thymulin on pituitary somatotropes is age dependent; Neu of the pharmaceutical composition are for treating chronic roendocrinology 1999, 69:20-27; Goya R G et al. Thymulin telogen effluvium, nonscarring alopecia, alopecia, hair loss, and the neuroendocrine system, Peptides 2004, 25:139-142). acute hair loss, non-scarring alopecia, balding. However, there is evidence that pituitary hormones them 0009. In another embodiment of the invention there is selves regulate thymulin secretion (Dardenne et al., Neuroen provided a pharmaceutical composition comprising an effec docrine control of thymic hormonal production. Prolactin tive amount of thymulin for stimulating human hair follicle stimulates in vivo and in vitro the production of thymulin by pigmentation and/or melanin granule production. human and murine thymic epithelial cells. Endocrinology 0010. In still further embodiments, the present disclosure 1989, 125: 3-12). provides a pharmaceutical composition comprising effective 0004) Thymosin alpha-1 (TA1) is a 28aa peptide, which amounts of thymulin and/or thymosin alpha-1 for maintain modulates lymphocyte differentiation and is involved in ing hair follicle cells longer in the anagen phase of the hair stimulating the production of antibodies and cytokines: cycle. endothelial cell migration, angiogenesis and wound healing (0011. In preferred embodiments, the present disclosure (Cordero et al., Thymic Peptides and Preparations: an contemplates that the effective amount of thymic peptides is Update; Arch Immun Therap Exp 1999, 47:77-82; Malinda in a formulation for topical application since such an appli KM et al. Thymosin alpha 1 stimulates endothelial cell cation usually results in less systemic side effects. A further migration, angiogenesis and wound healing; J Immunol preferred embodiment contemplates a composition wherein 1998, 160:1001-6). Further proposed therapeutic applica the effective amount of thymic peptides is in a formulation for tions encompass the enhancement of resistance to infections oral administration. In case of psychological strain, an intra and the treatment of viral infections and cancer (Hannappelet venous application of the formulation is contemplated. The al. The Thymosins. Prothymosin alpha, parathymosin, and effective concentrations and a preferred dosage can be taken beta-thymosins: structure and function; Vitamins and Hor from the examples, while a preferred dosage has still to be mones 2003, 66:257-296). found. It is contemplated that the thymic peptides of the 0005 Thymosin beta-4 (TB4) occurs in most human cells invention must be present in the composition in a concentra at high concentrations, with the highest concentration in tion for topical application in an amount from of 1 pg to 1000 platelets. Erythrocytes contain no thymosin B4 (HuffT et al., ng/mL, preferably 10 pg/ml to 100 ng/ml. Compositions for US 2011/028.1802 A1 Nov. 17, 2011
oral administration or by injection, intravenous, Subcutane mis (only partially) close to basal membrane using anti-thy ous, intramuscular or intra-peritoneal, will contain the thymic mosin beta-4 antibodies (dilution 1:500), 200-fold enlarge peptides for treatment of telogen effluvium, balding, hair loss, ment; alopecia in a dosage for administrationina range from 1 pMol 0029 FIG. 18 is a digital image on a photomicrograph to 1 nmol/kg. showing a immunohistological staining of a human hair fol licle (cytoplasm) using anti-thymosin beta-4 antibodies (dilu BRIEF DESCRIPTION OF THE DRAWINGS tion 1:500), 100-fold enlargement; 0030 FIG. 19 is a digital image on a photomicrograph 0012 FIG. 1 is a schematic representation of the experi showing a immunohistological staining of the proximal part mental design of assay #1; of a human hair follicle (the matrix cells in the pulp) using 0013 FIG. 2 is a schematic representation of the experi anti-thymosin beta-4 antibodies (dilution 1:500), 200-fold mental design of assay #2; enlargement; 0014 FIG. 3 is a schematic representation of the experi 0031 FIGS. 20a, b are digital images on photomicro mental design of assay #3; graphs showing a immunohistological staining of hair fol 0015 FIGS. 4A-4C are graphs showing the mean elonga licles for prolactin; tion in percent compared to day 0 effected by various con 0032 FIGS. 21a, b are digital images on photomicro centrations of thymulin, TA1 and TB4 in assay #1; graphs showing a immunohistochemical staining of hair fol 0016 FIG. 5 is a graph showing the mean elongation in licles for prolactin when treated with 100 pg/mL (a) and 10 percent compared to day 0 effected by thymulin and TB4 in ng/mL (b) thymulin, respectively; assay #2; 0033 FIG. 22A is a digital image on a photomicrograph showing a immunohistological staining of a human hair shaft 0017 FIGS. 6A-6C are graphs showing the mean elonga after acetone fixation using a rabbit polyclonal anti-thymosin tion in percent compared to day 0 effected by thymulin, TA1, antibody (dilution 1:100, lmmundiagnostik Ak 95.10), and TB4 in assay #3; 40-fold enlargement; 0018 FIG. 7 is a diagram showing the mean relative dark 0034 FIG. 22B is a digital image on a photomicrograph ening of the groups measured by image J obtained by a showing a immunohistological staining of a human hair shaft treatment with thymulin, TA1, and TB4; (root sheath) after acetone fixation using a rabbit polyclonal 0019 FIGS. 8A-8C are diagrams showing the proportion anti-thymosin antibody aa 75-109 (dilution 1:1000, Immun of hair follicles in different stages of the hair cycles for diagnostik Ak 9540), 40-fold enlargement; various hair follicle preparations in assays #1 and 3: 0035 FIG. 22C is a digital image on a photomicrograph 0020 FIG.9 is a diagram showing the mean percentage of showing a immunohistological staining of a human hair shaft Ki67-positive cells and the standard error of the measurement (root sheath) after acetone fixation using a rabbit polyclonal (SEM); anti-thymosin antibody aa 101-109 (dilution 1:200, Immun 0021 FIG. 10 is a digital image on a photomicrograph diagnostik Ak 9540), 40-fold enlargement; showing a immunohistological staining of human dermis 0036 FIGS. 23A,B are digital images on photomicro (Scalp skin) and epidermis using anti-thymulin antibodies graphs showing a immunohistological staining of human der (dilution 1:1000), 100-fold enlargement; mis and epidermis (scalp skin) and of a hair follicle using 0022 FIG. 11 is a digital image on a photomicrograph polyclonal rabbit anti-human thymosin antibodies (dilution showing a immunohistological staining of a human hair fol 1:100), 100-fold enlargement, and a corresponding control. licle (epidermis close to basal membrane) using anti-thymu lin antibodies (dilution 1:1000), 100-fold enlargement; DETAILED DESCRIPTION OF THE INVENTION 0023 FIG. 12 is a digital image on a photomicrograph showing a immunohistological staining of a hair shaft using 0037. The present disclosure relates to methods and com positions for treatment and prevention of human hair loss anti-thymulin antibodies (dilution 1:1000), 100-fold enlarge and/or hair greying. In particular, the present disclosure pro ment, vides composition comprising an effective amount of thymic 0024 FIG. 13 is a digital image on a photomicrograph peptides of the family thymulin, thymosin alpha-1 and thy showing a immunohistological staining of human dermis and mosin beta-4 and a pharmaceutical excipient, diluent or car epidermis (medulla, cortex and outer root sheath) using anti rier. In some preferred embodiments, the present disclosure thymosin-alpha-1 antibodies (dilution 1:500), 100-fold provides pharmaceutical composition comprising effective enlargement; amounts of thymulin and/or thymosin alpha-1 for maintain 0025 FIG. 14 is a digital image on a photomicrograph ing hair follicle cells longer in the anagen phase of the hair showing a immunohistological staining of a human hair fol cycle, and in particular, for treating hair shedding and telogen licle using anti-thymosin-alpha1 antibodies (dilution 1.500), effluvium by catagen inhibition. The disclosure therefore 100-fold enlargement; encompasses pharmaceutical compositions comprising 0026 FIG. 15 is a digital image on a photomicrograph thymulin and TA1 for treatment of chronic telogen effluvium, showing a immunohistological staining of the human hair nonscarringalopecia, alopecia, hair loss, acute hair loss, non shaft (inner rootsheath) using anti-thymosin alpha 1 antibod scarring alopecia, balding. In some preferred embodiments, ies (dilution 1:500), 100-fold enlargement; the disclosure provides pharmaceutical composition com 0027 FIG. 16 is a digital image on a photomicrograph prising an effective amount of thymulin for stimulating showing a immunohistological staining of the human dermis human hair follicle pigmentation and/or melaningranule pro and epidermis (outer root sheath) using anti-thymosin beta-4 duction. antibodies (dilution 1:500), 100-fold enlargement; 0038. While thymosin-beta and topically applied thymus 0028 FIG. 17 is a digital image on a photomicrograph extracts have long been claimed to stimulate hair growth in showinga immunohistological staining of epidermis and der rats, we note that it was never proven that they can stimulate US 2011/028.1802 A1 Nov. 17, 2011 human scalp hair growth. Therefore, we have explored ism, discontinuation of estrogen-containing medications; whether thymic peptides alter human hair growth and/or pig changes in diet like crash dieting, anorexia, low protein mentation in vitro. Instead, we have found that all tested intake, and chronic iron deficiency, heavy metals such as thymic peptides thymulin, Thymosin alpha-1 TA1, Thy Selenium, arsenic, and thallium. Acute telogen effluvium can mosin-beta4TB4) significantly inhibited hair shaft produc occur in either sex, but because hormonal changes in the tion of organ-cultured human scalp hair follicles (HF) under postpartum period are a common cause of telogen effluvium, serum-free conditions. However, thymulin or TA1-treated women may have a greater tendency to experience this con hair follicles stayed longer in anagen than vehicle-treated dition. In addition, womentend to find the hair shedding more control hair follicles. Nevertheless, TA1-treated hair follicles troublesome than men do so that more women seek medical showed fewer proliferating hair matrix keratinocytes than attention for this condition. Patients with acute telogen efflu control hair follicles. Quantitative Masson-Fontana his vium usually complain of relatively sudden onset of hair loss. tochemistry revealed further that thymulin-treated hair fol If greater than 25% of extracted hairs are in telogen, the licles had significantly more melanin granules than controls. TA1 and TB4 did not influence HF melanogenesis. Immuno diagnosis of telogen effluvium is confirmed. However, each histochemistry for all three thymic peptides appeared to show patient's scalp hair has an individual characteristic growth specific immunoreactivity in human Scalp skin sections. cycle. There are patients who have a very long anagen phase Thymulin and TA1 follow similar patterns with strongest and a small proportion of hair in telogen at any given time. immunoreactivity seen in epidermis and in the HF inner root Medications, of which the most frequency cited are beta sheath (especially Henle’s layer and medulla, distal to the hair blockers, anticoagulants, retinoids (including excess vitamin bulb). TB4 immunoreactivity was much more widespread, A), propylthiouracil (induces hypothyroidism), carbam and included also outer root sheath, dermal sheath and dermal azepine, and immunizations. Thus, this type of medication papilla of the HF. Only hair matrix keratinocytes appeared to represents itself as a problem. be negative for all three thymic peptides. Furthermore, we 0041. The hair is composed of a protein called keratin. The disclose that thymulin stimulates human hair follicle pigmen hair itself is arranged in three layers, an outer cuticle, middle tation. These combined data here provide first definitive evi cortex and central medulla. If the hair is coloured it is due to dence that thymic peptides alter human hair follicle biology, the presence of pigments—either melanin (black or brown) or and Suggest that thymulin and TA1 reduce telogen effluvium pheomelanin (red or yellow). If these pigments are lacking by catagen inhibition, even though actual hair shaft formation the hair is white. Canites is the term given to grey hair, it is an is also inhibited. illusion created by the mixture of white and coloured hairs. 0039 Telogen effluvium is a form of nonscarring alopecia Hair grows from a follicle. The walls of the follicle form the characterized by diffuse hair shedding, often with an acute outer root sheath of the hair. The lower part of the follicle onset. Telogen effluvium can affect hair on all parts of the body, but, generally, only loss of Scalp hair is symptomatic. widens out to form the hair bulb that contains the germinal Understanding the pathophysiology of telogen effluvium matrix, the source of hair growth. Dermal tissue projects into requires knowledge of the hair growth cycle. All hair has a the follicle base to form the dermal papilla, and this has a growth phase, termed anagen, apoptosis-driven regression network of capillary blood vessels to Supply oxygen, energy (catagen) and a resting quiescent phase, telogen (Paus et al., and the amino acids needed for growth. Melanocytes are The biology of hair follicles, NEJM 1999, 341:491-497). On present in the upper part of the papilla, producing pigment the scalp, anagen lasts approximately 3 years, while telogen granules that are distributed throughout the cortex. In the lasts roughly 3 months, although there can be wide variation follicle an inner rootsheath that has threelayers surrounds the between individuals. During telogen, the resting hair remains hair. The Henle’s layer is one cell thick and lies to the outer in the follicle until it is pushed out by growth of a new anagen root sheath. Huxley's layer is two or three cells thick and is in hair. In most people, 5-15% of the hair on the scalp is in the middle of the sheath. The cuticle of this inner root sheath telogen at any given time. Shedding does not occur until the interlocks with the cuticle of the hair. Both the hair and the new anagen hairs begin to grow. The emerging hairs help to inner root sheath grow at the same rate, but the inner root force the resting hairs out of the follicle. Recent evidence sheath breaks down about two-thirds of the way up the fol Suggests that the mechanism of shedding of a telogen hair is licle, so only the hair emerges past the skin Surface. Uncut an active process that may occur independent of the emerging hairs have a pointed tip. anagen hair. 0042. The present invention provides the first time a phar 0040. The symptom of both acute and chronic telogen maceutical composition that has immediate effect on the hair effluvium is increased hair shedding and diffuse hair loss shedding. While not promoting hair growth, the tested thymic from the entire scalp. Acute telogen effluvium is defined as peptides (thymulin, thymosin alpha-1, thymosin beta-4) sig hairshedding lasting less than 6 months. Patients usually only nificantly inhibited hair shaft production of organ-cultured complain that their hair is falling out at an increased rate or human scalp hair follicles (HF) under serum-free conditions, that the remaining hair feels less dense. Causes for telogen while interindividual variations were evident as expected, and effluvium and acute hair shedding can be physiologic stress, most interestingly, thymulin and/or TA1-treated hair follicles papulosquamous diseases of the Scalp Such as psoriasis and stayed longer in anagen phase than vehicle-treated control Seborrheic dermatitis, allergic contact dermatitis, immuniza hair follicles. Thus, thymic peptides can be used as an effec tions, severe infections (HIV), acute illness such as febrile tive ingredient in a pharmaceutical composition to inhibit illness, major Surgery and severe trauma as well as chronic telogen effluvium and hair shedding in both men and women. illness such as malignancy, particularly lymphoproliferative Most importantly, it has further been found that the applica malignancy, systemic lupus erythematosus, end-stage renal tion of thymulin leads to increased pigmentation and melanin disease, or liver disease, hormonal changes such as pregnancy granule production and is consequently an active ingredient and delivery (can affect both mother and child), hypothyroid against premature greying of hair. US 2011/028.1802 A1 Nov. 17, 2011
0043. In one embodiment, the present disclosure provides II. Isolation and Maintenance of Human Hair an easy to use topical therapeutic composition and treatment Follicles (Culture) for avoiding loss of hair. 0044. In another embodiment, the present disclosure pro 0052 Hair follicles were isolated as described (Philpott vides a therapeutic composition for topical application for MP et al, Human hair growth in vitro: J Cell Science 1990, increasing the lustre of hair as well as decreasing the greying 97(Pt 3):463-71). Isolated HFs were maintained in supple of hair. mented, serum-free Williams E medium (Biochrom, Cam 0045. A further embodiment in the present disclosure is bridge, UK) supplemented with 2 mMol/L L-glutamine (In represented by a treatment for hair loss through topical appli vitrogen, Paisley, UK), 10 ng/mL hydrocortisone (Sigma cation of thymic peptides in various formulations designed Aldrich, Taufkirchen, Germany), 10 ug/mL insulin (Sigma for topical application. Aldrich), and 1% antibiotic/antimycotic mixture (Gibco, 0046. Another embodiment is represented by a treatment Karlsruhe, Germany). Hair follicles were placed in a 24-well for hair loss through the oral application of thymic peptides in plate with 500 uL medium (day 0) and incubated in a CO2 various formulations designed for oral administration. incubator (5% CO2, 37°C.) over night without any treatment. 0047 Another aspect of the invention is represented by a The next day (day 1) medium was exchanged and test Sub treatment for hair loss and premature hair greying through the stance was added to each well (5 uL of Stock solution was oral application of thymic peptides, notably thymulin and added to 495 uL culture medium). The medium or medium TA1 in various formulations designed for oral application, with test Substance was changed every 48 hours. optionally combined with co-enzyme Q and acetyl carnitine. III. Experimental Design 0048 Still another aspect of the invention is represented by a composition and treatment for hair loss and hair greying 0053 Assay #1 Culture HF429: Hair follicles were grown through the topical application of thymic peptides, notably in the wells of a microtiter plate (3 follicles per well) and thymulin and TA1 in various formulations designed for topi treated for 9 days with culture medium and test substance as cal application, optionally combined with co-enzyme Q and indicated in FIG. 1. Thereafter, elongation, melanogenesis acetyl carnitine in various formulations designed for topical and hair cycle stage were determined. application. 0054 Assay #2 Culture HF 441: Hair follicle cultures (3 0049. The diagnostic aspect of the present invention is follicles/well) were treated for 9 days with culture medium represented by the use of antibodies against thymulin, TA1 and a combination of test Substances at various concentra and TB4 in a composition for diagnosis of telogen effluvium, tions as shown in FIG. 2, followed by a determination of upcoming alopecia, balding and hair loss. elongation and hair cycle stage. 0055 Assay #3 Culture HF 446: Eight hair follicle cul EXAMPLES tures (3 follicles/well) were treated with various concentra tion of test substance as described for 2 days, followed by 0050. This invention has been achieved by investigating Snap freezing and sectioning of two wells. Six wells were the effects of thymosin alpha-1 (TA1), thymosin beta-4 (TB4) treated up to 9 days, then Snap frozen and sectioned. The and thymulin on human hair follicles (HF) in vitro and by distribution of test Substances and concentrations were as studying and assessing the following parameters: elongation indicted in FIG. 3. The parameter measured after two days of hairshaft, melanogenesis/pigmentation, percentage of was proliferation/apoptosis, and the parameters measured cells proliferating and apoptosis and percentage of cells in after 9 days were elongation, proportion of cells in prolifera various hair cycle stages. The results have further been con tion/apoptosis, and hair cycle stage firmed by investigating and assessing the amount and distri 0056 Assay #4 Treatment of human scalp skin treated bution of thymic peptides inhuman scalp skin and human hair with antibodies against thymic peptides: Snap frozen human follicles, namely by immunohistochemistry and ELISA. Scalp skin sections of a 58 year old male (temporal area) were treated as follows: Inhibition of endogenous peroxidase with Example 1 3% H2O2 in TBS for 15 minutes, followed by three washings in TBS for five minutes, and pre-incubation with 10% goat I. Patients serum (Dako Cytomation, Glostrup, Denmark; Cat No 0051 Human anagen VI hair follicles were isolated from 501 14121) in TBS for 20 minutes, incubation with primary scalp skin obtained from three different individuals undergo antibody overnight at 4°C. (rabbit anti-Thymosin 1, anti ing routine face-lift Surgery. All experiments were performed Thymosin B4, anti-Thymulin, Immundiagnostik AG, Ben sheim, Germany CatNo's A9530.2, A9510.2, A9522.2 according to Helsinki guidelines. respectively; anti-Thymosin 1 and anti-Thymosin B4 diluted 1:500 in TBS comprising 2% goat serum (DakoCytomation). TABLE 1. The anti-Thymulin was diluted 1:1000). A further washing in Details of patients TBS followed and incubation with secondary antibody (goat anti-rabbit-biotinylated, Jackson Immunoresearch, PA,USA: Name of Age CatNo 111-065-045), diluted 1:200 in TBS comprising 2% sample Sex Localisation (years) goat serum for 45 minutes at room temperature. A treatment Assay #1 HF 429 f occipital 69. with avidin/biotin complex-peroxidase (Vector Laboratories, Assay #2 HF 441 f occipital 44J. Assay #3, #5a, #5b and HF 446 f occipital 61 Burlingame, Calif., USA; CatNo PK-6100) for 30 minutes Assay #4 HS 336 l temporal S8. followed and then a further washing in TBS. Thereafter treat Assay #5c HS 366 f temporal 58 ment with AEC+substrate (Vector Laboratories, Burlingame, Calif., USA; CatNo SK-4200) for 5 minutes and washes in TBS. Counterstaining was done with Mayer's Haemalaun US 2011/028.1802 A1 Nov. 17, 2011
(Chroma Muenster, Germany; CatNo 2E038) and mounting 30 minutes, then washes in TBS. Thereafter treatment with with Aquamount (DAKO Cytomation, CatNo S3025). Fast Red Tablets (Sigma-Aldrich, CatNo F4648-50SET) for 6 0057 Assay #5—ELISA—amount of thymic peptides in minutes. Counterstaining with Mayer's Haemalaun (Chroma human hair follicles: Three samples were tested: Assay 5a: 20 Muenster, CatNo 2E038) and mounting with Aquamount Hair follicles from the occipital area of a 61 yr old female (DAKO Cytomation, CatNo S3025). patient; Assay 5b: 20 Hair follicles from same location and of 0061. In both assays human pituitary gland was set as same female patient as in 5a; Assay 5c: 20 Hair follicles from positive control and negative control were hair follicles the temporal area of a female patient (58 yr). The twenty treated without the primary antibody. dissected hair follicles were immersed in phosphate buffer comprising 1% (w/v) TRITONTM X (Fluka, Sigma Aldrich, Taufkirchen, Germany; CatNo 93.420). 550D1 PBS was IV. Measurement of Elongation added and the samples were emulsified by ultrasonic disinte gration and centrifugation. The Supernatant was used in the 0062 Hair shaft length measurements were performed ELISA. A sample of the diluted antigen Thymulin, Thymosin every second day on individual hair follicles using a binocular C1 and Thymosin B4 (all Immundiagnostik AG, Bensheim, microscope with an eyepiece measuring reticule. The length Germany, CatNos. A 9530AG.1. A 9510AG1 and A9522AG. of the follicle was defined as the distance between the base of 1, respectively) were used as positive control. The thymulin, the bulb and the cut end of the hair fibre (Philpott MP et al. thymosin C.1 and thymosin 34 were determined using com Human hair growth in vitro: J Cell Science 1990, 97(Pt mercial ELISA kits (Thymulin EIA CatNoK9810; Thy 3):463-71). mosin C.1 ELISA CatNo K95.10: Thymosin B4 EIA CatNo K9520 all Immundiagnostik AG, Bensheim, V. Measurement of Melanogenesis Germany). 0058 Assay #6. Stimulation of expression of pituitary 0063 Fontana Masson staining was performed at the end hormones by Thymulin (PRL+TSH): Hair follicles from the of the culture period and photos taken of snap frozen sections. occipital area of a 61 year old female were cultured for 9 days The photos were analysed (Image J. public domain, devel and treated with culture medium and thymulin 10 pg/mL oped at the National Institutes of Health, USA) by measuring (human thymulin, No A9530AG.1. Immundiagnostik AG, the density of two squares, consisting of 150x150 dots, placed Bensheim, Germany) as described above, then Snap frozen over the darkest areas on both sides of the dermal papilla. For and sectioned. The staining was performed according to the Fontana-Masson staining, fixed sections were treated in dark following protocol: ness for 40 minutes with a solution containing 10% silver 0059 A. Prolactin: Pre-treatment with 0.5% Triton X nitrate (Merck & Co., Inc., Whitehouse Station, N.J., USA; (Fluka, Sigma Aldrich, Taufkirchen, Germany; CatNo CatNo 1.01512.01.00), following by a development in solu 93.420) in TBS for 10 minutes at room temperature, three tion containing 5% sodium thiosulphate (Merck & Co., Inc.; washings in TBS for five minutes each, treatment with 0.6% CatNo 1.06512.2500) for 1 minute and multiple washings in H2O in methanol three times for 15 minutes, washes in TBS. distilled water for 3 minutes. Counterstaining with 0.5% Neu Pre-incubation with 10% rabbit serum (DakoCytomation, tralrot (Chroma Muenster, Germany, CatNo 2E038). Then CatNo X0902) in TBS with 3% BSA for 20 minutes, incuba dehydration in xylol and covering with Eukitt (O. Kindler tion with primary antibody (goat anti-human prolactin, Santa GmbH, Freiburg, Germany; CatNo E115). Cruz, Biotechnology, Inc., Santa Cruz, Calif., USA; CatNo SC-7805), dilution 1:100 in TBS+2% rabbit serum (Dako VI Assessment of Percentage of Proliferating Cells Cytomation, CatNo X0902) overnight at 4° C. Thereafter and Cells Undergoing Apoptosis washes in TBS. Incubation with secondary antibody (rabbit anti-goat-biotinylated, DAKO Cytomation, CatNo E0466) 0064. After nine days, Ki67/TUNEL staining was per diluted to 1:200 with 2% rabbit serum in TBS for 45 minutes formed and photos taken. at room temperature. Treatment with Avidin Biotin Complex 0065 Proliferating and resting cells as well as cells under alkaline phosphatase (Vector Laboratories, Burlingame, going apoptosis were counted separately proximal Auber's Calif., USA; CatNo AK-5000) for 30 minutes, then washing line. Results are expressed as percentages. For Ki-67/TUNEL in TBS. Thereafter treatment with Fast Red Tablets (Sigma staining, fixed sections were incubated for 60 minutes in a Aldrich, CatNo F4648-50SET) for 6 minutes. Counterstain solution comprising 30% TaT-enzyme (Chemicon Interna ing with Mayer's Haemalaun (Chroma Muenster, CatNo tional, Inc., Temecula, Calif., USA; CatNo S1707).2 mL stop 2E038) and mounting with Aquamount (DAKO Cytomation, buffer (Chemicon International, Inc.; CatNo S7110), diluted CatNo S3025). in 68 mL distilled water, was added for 10 minutes to stop the 0060 B.TSH: Pre-incubation with 10% goat serum (Dako reaction. This was followed by a pre-incubation for 20 min Cytomation, CatNo 501 14121) in TBS for 20 minutes, then utes with 10% goat serum (Dako Cytomation, CatNo incubation with primary antibody (mouse anti-human TSH, 501 14121) and incubation with mouse-anti human Ki-67 Dianova, Hamburg, Germany, CatNo DLN 07830), dilution antigen (Chemicon International, Inc., CatNo S7110) 1:20 in 1:20 in TBS+2% goat serum (Dako Cytomation, CatNo PBS with 2% goat serum. The fixed section was then washed 501 14121) overnight at 4°C. Thereafter washing in TBS. in PBS, treated with anti-digoxigenin antibody (Chemicon Incubation with secondary antibody (goat anti-mouse-bioti International, Inc., CatNo S71 10) (59DI Antibody solution nylated, Beckman Coulter, Inc. Fullerton, Calif., USA, and 5601 Stop solution, 30 minutes) and goat anti-mouse CatNo IM0816) diluted to 1:200 with 2% goat serum in TBS IgG-rhodamine red (Jackson Immuno Research, Suffolk, for 45 minutes at room temperature. Treatment with avidin/ UK; CatNo 115-295-062), 1:200 in PBS with 2% goat serum. biotin-complex coupled with alkaline phosphatase (Vector After washes with PBS, counterstaining with DAPITM (10 Laboratories, Burlingame, Calif., USA; CatNo AK-5000) for g/mL) (Roche, Basel, Switzerland; CatNo 236276) and fur US 2011/028.1802 A1 Nov. 17, 2011
ther washes with PBS and mounting using Fluoromount (Southern Biotech, Birmingham, Ala., USA; CatNo 0100 TABLE 2 01). Mean elongation with thymulin in percent compared to day O VII. Assessment of Hair Cycle Stage Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 0066 Photodocuments from the Fontana Masson staining Control O 17.38 34.92 62.57 8OSO 94.86 Thymulin O 13.19 37.58 64.05 75.10 90.98 were used for hair follicle staging according to previously 10 pg/mL. well-defined morphological criteria (StennK Setal, Controls Thymulin O 11.33 31.50 53.75 68.76 83.13 of Hair Follicle Cycling: Physiol Rev 2001, 81:449-494, 100 pg/mL. Müller-Röver Set al. A Comprehensive Guide for the Accu SEM: rate Classification of Murine Hair Follicles in Distinct Hair Cycle Stages; J Invest Dermatol 2001, 1 17:3-15). The per Control O.OO 186 2.64 2.55 4.21 S.O2 centage of hair follicles in anagen, early, mid and late catagen Thymulin O.OO 3.17 4.11 4.13 4.01 3.36 was determined. 10 pg/mL. Thymulin O.OO 186 3.11 3.45 1.69 3.SO 100 pg/mL. VIII. Statistical Analyses
0067 All data were analysed by two-tailed unpaired t-test TABLE 3 (SPSS analysis software SPSS Inc., Chicago, USA). For determination of elongation, two outliers at top and bottom Mean elongation in percent compared to day 0. end of value range were eliminated; with the measurement of Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 melanogenesis one outlier at both, top and bottom range, was Control O.OO 17.38 34.92 62.57 80.SO 94.86 eliminated. Results are given as mean value and standard Thymosin alphc.1 O.OO 16.71 34.54 S1.14 70.11 82.SS error of measurement (SEM). 100 ng/mL Thymosin alphc.1 O.OO 1541. 34.74 SO.66 66.20 75.26 1000 ng/mL Results SEM:
0068 Microdissected anagen VI hair follicles of three Control O.OO 1.86 2.64 2.55 4.21 S.O2 independent experiments were treated with medium only or Thymosin alpha 1 O.OO 2.75 3.62 3.67 4.46 5.33 100 ng/mL thymulin, TA1 or TB4 at various concentrations. Elongation Thymosin alpha 1 O.OO 1.94 2.83 5.56 5.95 7.04 was measured every two days and growth ratio was calcu 1000 ng/mL lated. Elongation of different groups was compared to each other and the elongation results were analysed using Stu dent's t-test. Treatment by TA1, TB4 and thymulin gave no TABLE 3 statistical significant prolonged hair growth. Instead, treat ment with TB4 (100 ng/mL and 1000 ng/mL). TA1 (100 and Mean elongation in 90 compared to day 1 1000 ng/mL) and thymulin (100 pg/mL) lead to a signifi Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 cantly reduced hair growth compared with the control, while Control O.OO 17.38 34.92 62.57 8OSO 94.86 substantial interindividual differences in the HF response to Thymosin beta4 0.00 12.26 35.49 49.79 61.36 74.11 thymic hormones was observed. 100 ng/mL Thymosin beta4 0.00 14.2O 39.31 66.2O 90.23 103.17 0069. In the first experiment (assay #1) the relative elon 1000 ng/mL gation of the groups was between 74.11 and 103.17% at day SEM: 9. Follicles treated with thymosin B4100 ng/mL and thymu Control O.OO 1.86 2.64 2.55 4.21 S.O2 lin 100 pg/mL showed a significant lack of hair growth (p<0. Thymosin beta4 0.00 1.34 2.OO 2.16 2.13 2.11 05). In the second experiment (assay #2) only two Substances 100 ng/mL Thymosin beta4 0.00 2.30 2.13 1.67 3.22 3.63 were tested, one only in a single concentration. We were not 1000 ng/mL able to detect any significant differences of relative hair growth. The elongation of the groups was between 43.30 and 58.90%. In a third experiment (assay #3) the relative elonga 0070 Results obtained in Assay 2 are shown in Tables 4 to tion of the groups was between 69.90 and 94.36%. The treat 7 and in FIG. 5. ment with thymosin C.1 at 100 and 1000 ng/mL and thymosin B4 at 1000 ng/mL produced a significantly lowered rate of TABLE 4 hair growth (p<0.05). The differences between the results of Mean elongation in 96 compared to day O: assays #1, #2 and #3 also point to interindividual variations in the response of human scalp hair follicles to thymic peptides. Day O Day 3 Day 5 Day 7 Day 9 The results as to the mean elongation in percent compared Control O 21.01 32.30 41.90 48.79 with day 0 are graphically summarized in Tables 2 to 4 below Thymulin 10 pg/mL O 20.86 34.97 43.26 S8.90 and FIGS 4A-4C US 2011/028.1802 A1 Nov. 17, 2011
TABLE 4-continued TABLE 5-continued Mean elongation in 96 compared to day O: Mean relative darkening of the groups (measured by image
Thymosin beta4 O 1935 3166 41.77 49.94 Mean SEM 100 ng/mL Thymosin beta4 O 21.68 32.59 37.48 43.30 TB41000 ng/mL 85.52 4.53 1000 ng/mL Thymulin 10 pg/mL 75.73 6.68 Thymulin 100 pg/mL+ 90.48 7.68 SEM: Day 1 Day 3 Day 5 Day 7 Day 9 0073 Statistical analysis shows that there was signifi cantly more staining in the thymulin 10 pg/mL-treated group Control O 1.45 2.33 3.54 4.OO Thymulin 10 pg/mL O 2.16 2.38 2.26 2.81 than in the control group. A mean darkness (brightness) value Thymosin beta4 O 1.58 1.60 1.60 2.02 of 75.73 indicates therefore that this portion contained rela 100 ng/mL tive more melanin pigment. Thymosin beta4 O 1.47 1.72 1.55 2.22 1000 ng/mL 3. Influence of Thymic Hormones on Hair Cycle Stage: Thymulin and TA1 Seem to Keep HFs in Anagen (Assays #1, 0071. The mean elongation in percent compared to day 0 #2 and #3) in assay #3 obtained by thymulin, TA1 and TB4 are shown in 0074 Photodocuments from the Fontana Masson staining Table 4 and FIGS. 6A-6C. were used for hair follicle staging according to previously well-defined morphological criteria (Stenn KS etal, Controls TABLE 4 of Hair Follicle Cycling: Physiol Rev 2001, 81:449-494, Mean elongation in 90 compared to day 0 Müller-Röver Set al. A Comprehensive Guide for the Accu rate Classification of Murine Hair Follicles in Distinct Hair Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 Cycle Stages; J Invest Dermatol 2001, 117:3-15). The per Control O 6.23 42.68 62.47 76.66 86.77 centage of hair follicles in anagen, early, mid and late catagen Thymulin 10 pg/mL O 7.30 45.35 58.41 67.70 78.70 was determined in the different assays. Comparing the three Thymulin 100 pg/mL 0 5.86 40.11 62.88 80.67 94.36 different assays it is evident that groups treated with Thymu TA1 100 ng/mL O 6.25 45.55 49.52 61.94 69.90 lin 10 pg and 100 pg/mL have consistently a higher propor TA1 1000 ng/mL O 4.OO 37.95 51.77 68.36 73.33 TB4 100 ng/mL O S.91 42.SS 59.58 73.62 83.13 tion of HFS in anagen than control groups. TB4 1000 ng/mL O 8.28 39.17 53.52 6S.S1 70.08 (0075 Groups treated with TB4100 ng have in both assays SEM: tested (Assay #1 and #3) a higher proportion of cells in Control O O.S9 1.55 2.17 2.82 3.76 catagen compared to control. The proportions of hair follicles Thymulin 10 pg/mL O O.70 1.57 2.39 2.63 2.73 in different stages of the hair cycle are shown in FIGS. 8a-c. Thymulin 100 pg/mL 0 O.9S 2.05 2.31 2.19 2.37 It should be noted that anagen is the stage ofhair growth in the TA1 100 ng/mL O O.80 5.95 3.16 4.09 5.25 hair cycle, that hair follicles cease to grow for a short period TA1 1000 ng/mL O O.S4 1.11 1.73 3.26 3.94 TB4 100 ng/mL O O.66 1.23 2.13 2.36 2.28 when they reach the stage of catagen (marked by morpho TB4 1000 ng/mL O 1.06 1.16 1.92 2.32 3.03 logical changes that accompany this change) and that they then enter the stage of telogen, which is the resting stage of the hair follicle. Once the hair follicle enters the stage of catagen 2. Treatment with Thymulin Leads to Significantly More the growth of hair is finished and it will fall out. Consequently, Melanin (Assay #1) FIGS. 8a-c show the percentage of hair follicles in the differ 0072 Micro-dissected anagen VI hair follicles were ent stages of human hair cycle after a 9-day treatment with the treated as above described. At the end of the culture period three Substances in two concentrations each. The results Fontana Masson staining was performed and photos were therefore support that in all three assays there were more taken of the Snap frozen sections. The pictures were analysed follicles in both thymulin-treated and both thymosin alpha-1 by measuring the darkness (brightness) of two squares, con treated groups in anagen than in the control groups. This sisting of 150x150 dots, placed over the darkest areas on both shows therefore that both thymulin and thymosin alpha-1, by sides of the dermal papilla (dark-small index number, keeping human hair follicles longer in the stage of anagen, light-high index number). The shading of the different prevent the hair follicle from entering the stage of catagen and groups was compared to each other and the results were that both thymulin and thymosin alpha-1 can therefore pre analysed using Student's t-test. The results are shown below vent from hair loss. in Table 5 and FIG. 7. 4. More Precisely, Human Hair Follicles Treated for Nine Days with Thymosin C.1 100 ng/mL and 1000 ng/mL Exhib TABLE 5 ited a Significantly Reduced Percentage of Proliferating Cells Compared to Control (Assay #3) Mean relative darkening of the groups (measured by image 0076. After nine days, Ki67/TUNEL staining was per Mean SEM formed and photos were taken. In anagen hair follicles, pro liferating cells and cells undergoing apoptosis were counted Control 95.26 8.10 Thymosin C.1 100 ng/mL 81.01 5.57 separately proximal Auber's line. Results are expressed as Thymosin C.1 1000 ng/mL 71.61 9.36 percentages. Statistical analysis show a significant (p<0.05) TB4100 ng/mL 101.04 6.75 less percentage of cells are proliferating in the groups treated with thymosin-C.1 100 ng/mL and thymosin-C.1 1000 ng/mL US 2011/028.1802 A1 Nov. 17, 2011
compared to the control. The group with hair follicles treated abundant evidence that thymosin alpha 1 or at least thymosin for two days only proved to be too small to make a statistical like peptides have a physiological role in the control of the analysis. The results are summarized in Table 6 and FIG. 9 hair growth and cycle. showing the mean percentage of Ki67 positive cells with SEM 6. Thymulin May Stimulate Human Hair Follicles to Produce Pituitary Hormones (Assay #6) TABLE 6 007.9 These very preliminary results indicate that Thymu Mean percentage of proliferating cells lin might stimulate the hair follicles to produce Prolactin but after 9 days of culture, Anagen only there is no indication that this is true for TSH, too. However, the slides, which were the “lower quality” slides with partly Mean percentage incomplete follicles from the other tests, suffered visibly of Ki67-positive from the initial culturing and the following staining method. cells SEM The immunoreactivity for Prolactin in the untreated follicle Control 45.12 3.84 was in the area in between IRS and ORS, in keeping with TA1100 ng/mL 25.15 6.35 previous findings (Foitzik Ket al: Human Scalp Hair Fol TA11000 ng/mL 28.05 3.33 TB4100 ng/mL 39.30 6.2O licles Are Both a Target and a Source of Prolactin, which TB41000 ng/mL 33.14 4.45 Serves as an Autocrine and/or Paracrine Promoter of Apop Thymulin 10 pg/mL 31.58 6.OO tosis-Driven Hair Follicle Regression: Am J Pathol 2006, Thymulin 100 pg/mL 35.75 9.04 168:748-756). In few hair follicles stimulated with Thymulin 10 pg/ml and 100 pg/ml there was some additional staining in the IRS. 5. Immunohistochemical Staining of Thymic Hormones in Human Scalp Skin and Human Hair Exhibits Stainings in a The Immunoreactivity for TSH Did not Show any Differ Specific Looking Pattern (Assay #4) ences in Between Thymulin-Treated and Control Hair Fol licles. 0077. As evident in the pictures, all antibodies to thymic 0080 Again one must stress, that these investigations hormones seem to find binding places in the human skin in a were made with only few slides, to few to risk a statement specific pattern. Thymulin staining is most impressive in the about whether Thymulin is able to stimulate the production of epidermis, especially close to the basal membrane (FIG. 11). pituitary hormones in hair follicles. Regarding the hair follicle, the inner root sheath, especially 7. ELISA Showed that Thymulin and TA1 are Present in Henle’s layer is stained. However, medulla, cortex and outer Micro-Dissected Human Hair Follicles (Assay #5) root sheath (minimal, close to IRS) have also acquired some I0081. Thymulin was detected in two of the three samples. staining (FIG. 12 and FIG. 13). It is noticeable that the stain ing starts where the hair bulb merges into the shaft, the matrix The third sample was invalid. cells are not stained. The staining for thymosin alpha1 follows Sample #5a invalid the same pattern: strong staining in the epidermis, strongest at Sample #5b 0.157 ng/mL the basal membrane (FIG. 14) and staining of the IRS begin Sample #5c O.089 ng/mL TA1 was detected in all three ning where the bulb ends (FIG.15 and FIG.16). There is little samples with the following concentrations: staining of medulla and cortex but no staining of the ORS Sample #5a 2.604 ng/mL (extrapolated data) Sample #5b (contrary to anti-Thymulin). The staining for TB4 stains more 5.003 ng/mL ubiquitary than the previous two. There is heavy staining of Sample #5c O.493 ng/mL (extrapolated data) the dermis, especially the epidermis close to the basal mem Thymosin B4: all samples were invalid brane (FIG. 17) in a cytoplasmatic pattern (FIG. 18). In the Note: Samples #5a and #5b were from the same individual hair the staining is strongest in the IRS but is present also in ORS, medulla, dermal sheath and dermal papilla (FIG. 19). Summary Interestingly the only place with no staining is the area of 0082. These studies showed that all three substances have matrix cells in the bulb (FIG. 20), an area not stained with different influences on human hair follicles in vitro, while anti-Thymulin and anti-thymosin alpha 1, either. there are interindividual differences in the HF response to 0078 FIG. 22 A-C show immunohistological examina thymic hormones. tions of the scalp and hair bulb for endogenous thymosin-like I0083) Thymulin did show different results regarding hair peptides with or without acetone fixation of the object and shaft elongation in all three assays. It seemingly tended to using different antisera for thymosin peptides. FIG. 22A elongate the hair shaft in assay #2 and inhibited elongation shows an immunostaining of the hair shaft for thymosin-like significantly in assay #1. The content of melanin was signifi peptides using a polyclonal anti-thymosin alpha 1 antibody cantly raised in the HFS treated with thymulin 10 pg/mL. from rabbit (Immundiagnostik AG, Bensheim, DE-Ab Highly interesting is the fact that there are consistently more CatNo. 95.10); FIG. 22B of the inner root sheath using a hair follicles in the hair cycle stage of anagen after treatment site-specific anti-thymosin alpha 1 antibody against amino with thymulin than in the control groups. This result was acids 75-109 (Immundiagnostik AG, Bensheim, DE-Ab consistent in all three individuals tested. In these anagen-hair CatNo. 9540); FIG.22C of the inner root sheath using a site follicles the thymulin-treated groups showed a lower percent specific anti-thymosin alpha 1 antibody against amino acids age of proliferating cells than control (only one individual 101-109 (Immundiagnostik AG, Bensheim, DE-Ab CatNo. tested), but not to a significant level. Immunohistochemistry 9570) FIG.23A, B show a section and histological staining of for thymulin showed staining mainly in the IRS (Henle’s human dermis and epidermis using anti-thymosin alpha 1 layer), medulla and cortex with sparing of the hair matrix cells antibodies. The immunohistological examinations contain and dermal papilla. The ORS shows only minimal staining. US 2011/028.1802 A1 Nov. 17, 2011
The human Scalp skin showed staining mainly in the epider catagen. Groups of both peptides with all tested concentra mis, strongest at the basal membrane. These tests indicate that tions had in all three assays a higher percentage of hair fol thymulin stimulates human hair follicles to express prolactin. licles in anagen than the control group after 9 days of treat We could not show the same for TSH. ELISA showed that the ment. One has to speculate by which mechanisms this is human hair follicle contains thymulin. achieved. We could not find such a striking correlation in 0084 Thymosin alpha1 inhibited in both assays hair shaft between a “higher than in the control group' number of elongation in either concentration, in assay #3 on significant follicles in anagen and Thymosin B4. Interestingly, this is the level (both concentrations). We could not find any influence only one of the tested peptides where such a correlation has on melanogenesis with the here applied methods. After 9 days been made (Philp D et al. Thymosin beta4 increases hair of treatment there were more hair follicles in anagen in the growth by activation of hair follicle stem cells: The FASEBJ thymosin alpha1-groups than in control, this applied to both Express Article doi:10.1096/f.03-0244fe Published online tested individuals and all concentrations. On examination of Dec. 4, 2003). However, our results don't stand in contrast to these anagen follicles there were significantly less cells pro these findings as the TB41000 ng/mL-treated group has in all liferating than in the control group (both concentrations). three assays the same or more hair follicles in anagen. The Interestingly, we noticed exactly the same trend with the difference to the control group is just not as impressive as with thymulin-treated groups. Staining for Thymosin alpha1 the other peptides. Interestingly, all Substances showed a showed a picture similar to the one for thymulin-staining of lower share in proliferating cells in these anagen-hair follicles the epidermis, strongest close to the basal membrane. The than the control group (only thymosin alpha 1 to a significant hair follicle shows the heaviest staining in the IRS beginning level, a fact that could explain the inhibiting effect on elon with the hair shaft and there is no staining of hair matrix cells, gation. dermal papilla and ORS (contrary to thymulin). The ELISA I0087 We have shown an influence of thymulin on the confirmed the presence of thymosin alpha1 in the human hair content of melanin in the hair follicle in the one assay inves follicle. tigated. This result might be of significant clinical importance 0085 TB4 inhibited hair growth significantly in two out of and it is a completely new finding, which has not been the six organ cultures. This happened with both concentra described before. It certainly warrants further research to find tions tested but not consistently throughout all tests. Indeed in out about the underlying mechanism. Furthermore additional Assay #1 there was some increased elongation in the follicles tests of Melanogenesis should be applied which may disclose treated with a concentration 1000 ng/mL, but not to a signifi further clues about the influence of thymic peptides on Mel cant level. However, the same concentration showed in Assay anogenesis. The ELISA and immunohistochemistry con #3 significantly delayed elongation. We could not find any firmed the presence of thymic peptides in the hair follicle and influence on melanogenesis, hair cycle stage and amount of human Scalp skin although it remains to be confirmed that the proliferating cells in our assays with the methods applied. staining is specific for the tested thymic peptide. Thymosin B4-treated cells did not show any massive differ 0088. In view of these results, it is therefore obvious to ences in hair cycle stage in our assays, but it is noticeable, that repeat hair cultures with lower concentrations of TB4 (1 there are consistently more cells in the stage of anagen in the ng/mL and 10 ng/mL) and to perform further tests of melano group treated with 1000 ng/mL after nine days in the than in genesis (such as counting the active melanocytes etc.) on the the controls. Hair follicles in anagen, treated with either of the already collected data and finally to repeat the hair culture both concentrations of TB4 had less proliferating cells than with hair from another individual to establish if the influence control but not to the significant level. Staining for TB4 of Thymulin on melanogenesis can be repeated. Furthermore, showed heavy staining of the dermis, especially the epidermis it was noted that GH-treated hair follicles stained more inten close to the basal membrane in a cytoplasmatic pattern. In the sively for thymulin than the control group as GH stimulates hair the staining is strongest in the IRS but is present also in the secretion of thymulin (Timsit J et al. GH and IGF-1 ORS, medulla, dermal sheath and dermal papilla. The only stimulate hormonal function and proliferation of thymic epi place with no staining is the area of matrix cells in the bulb. thelial cells; J Clin Endocrinol Metab 1992, 75:183-8). The ELISA failed with both assays. I0086) Even though we could not confirm a striking and 1.-15. (canceled) desired effect of the tested thymic peptides on hair shaft 16. A method for the treatment or prevention of human hair elongation in vitro we certainly have proven that all three loss and/or hair graying, comprising administering to a proteins significantly influence different components of the patient Suffering from hair loss and/or hair graying an effec hair and of the hair cycle. Contrary to our expectations raised tive amount of one or more thymic peptides of the families of by reports from Philp et al. (The FASEB J Express Article thymulin, thymosin alpha-1 and thymosin beta-4, and a phar doi:10.1096/f.03-0244fe Published online Dec. 4, 2003) maceutical excipient, diluent or carrier. and Melinda K Metal, Thymosin alpha 1 stimulates endot 17. The method as claimed in claim 16, comprising admin helial cell migration, angiogenesis and wound healing; J istering an effective amount of thymulin. Immunol 1998, 160:1001-6) not a single peptide tested 18. The method as claimed in claim 16, comprising admin improved significantly the elongation of the cultured hair istering an effective amount of thymosin alpha-1. follicles. Indeed, in a few cases there was significant reduc 19. The method as claimed in claim 16, comprising admin tion of the growth rate. Overlooking the (very limited) studies istering an effective amount of thymosin beta-4. it appears that speeding up the elongation is not the mecha 20. The method as claimed in claim 16, comprising admin nism by which the peptides improved hair growth in the istering effective amounts of thymulin and thymosin alpha-1, previous mentioned studies. A possible explanation for this and a pharmaceutical excipient, diluent or carrier. paradox could lie in the observation that both, thymulin and 21. The method as claimed in claim 16, wherein the treat thymosin alpha1-treated hair follicles seem to prolong the ment maintains hair follicle cells longer in the anagen phase stage of anagen and/or prevent hair follicles from entering of the hair cycle. US 2011/028.1802 A1 Nov. 17, 2011
22. The method as claimed in claim 16, wherein the hair 30. A pharmaceutical composition for the treatment and loss is related to hair shedding and telogen effluvium by prevention of human hair loss and/or hair greying, comprising catagen inhibition. an effective amount of one or more thymic peptides of the 23. The method as claimed in claim 16, wherein the hair families of thymulin, thymosin alpha-1 and thymosin beta-4, loss is related to chronic telogen effluvium, nonscarring and a pharmaceutical excipient, diluent or carrier. alopecia, alopecia, hair loss, acute hair loss, non-scarring 31. The pharmaceutical composition as claimed in claim alopecia or balding. 30, comprising an effective amount of thymulin and thymosin 24. The method as claimed in claim 16, wherein the treat alpha-1. ment stimulates human hair follicle pigmentation and/or 32. The pharmaceutical composition as claimed in claim melanin granule production. 30, for topical application. 25. The method as claimed in claim 16, wherein the effec 33. The pharmaceutical composition as claimed in claim tive amount of one or more thymic peptides is administered 32, in the form of an hydrogel. topically. 34. The pharmaceutical composition as claimed in claim 26. The method as claimed in claim 25, wherein the pep 30, for oral or systemic administration. tides are administered in doses having a concentration rang 34. A method for diagnosis of telogen effluvium, balding, ing from about 1 pg/mL to about 1000 ng/mL. hair loss or alopecia, comprising contacting a sample of skin 27. The method of claim 25, wherein the peptides are or comprising hair follicles with and antibody against thymu administered in doses having a concentration ranging from lin, thymosin alpha-1 or thymosin beta-4, and quantitating the about 10 pg/mL to about 100 ng/mL. amount of thymulin, thymosin alpha-1 or thymosin beta-4 in 28. The method as claimed in claim 16, wherein the effec said sample. tive amount of one or more thymic peptides is administered 35. A kit for diagnosis of telogen effluvium, balding, hair orally or systemically. loss or alopecia, comprising containers of antibodies against 29. The method as claimed in claim 28, wherein the pep thymulin, thymosin alpha-1 and thymosin beta-4. tides are administered in doses ranging from about 1 pmol/kg to about 1 nmol/kg. c c c c c